Horseshoe crab amebocyte lysate factor G subunit A

ABSTRACT

This invention relates to a DNA shown by SEQ ID No. 1 and an amino acid sequence coded by said DNA. This invention also relates to a DNA and an amino acid sequence of subunit a of (1→3)-β-D-glucan sensitive factor derived from amebocytes of horseshoe crab and have potent affinity to (1→3)-β-D-glucan in cell walls of fungi. Therefore, the invention is useful for the diagnosis of fungal diseases and as antimicrobial or eradicating agent of fungi in combination with an antifungal agent.

FIELD OF THE INVENTION

This invention relates to a polypeptide derived from limulus (horseshoe crab) amebocytes shown by an amino acid sequence of (1→3)-β-D-glucan sensitive factor (hereinafter abbreviated as factor G) and a DNA encoding thereof, particularly to a polypeptide shown by an amino acid sequence of subunit a of factor G and a DNA encoding thereof.

BACKGROUND OF THE INVENTION

Heretofore, determination of endotoxin with a limulus amebocyte lysate has been known. This method is based on coagulation of lysate with a very small amount of endotoxin, for example at about 10⁻⁹ g. It has been elucidated with the progress of biochemistry that this coagulation reaction consists of the stepwise activation mechanism of some coagulation factors. (Takanori Nakamura et al., Jpn. J. Bacteriol., 38, 781-803 (1983)). FIG. 1 shows the coagulation cascade reaction of Japanese horseshoe crab (Tachypleus tridentatus). The structures of three serine protease precursors (factor C, factor B and proclotting enzyme) and a clottable protein (coagulogen) in FIG. 1 have been elucidated by studies such as cDNA cloning (T. Muta et al. (1991) J. Biol. Chem., 265, 22426-22433 and 266, 6554-6561, and T. Miyata et al (1986) J. Biochem., 100, 213-220).

The lysate has been known to react with (1→3)-β-D-glucan which exists in the cell walls of fungi and yeasts, at concentrations of 10⁻⁸ -10⁻⁹ g to trigger coagulation (see FIG. 1). Factor G responds to (1→3)-β-D-glucans, initiating clot formation. It is reported that factor G is a serine protease precursor as well as the other factors and it is a glycoprotein composed of non-covalently associated subunits a (72 kDa) and b (37 kDa) (64th Annual Meeting of Japan Biochemical Society, 1991).

Additionally, subunit a of factor G has a specific binding site to (1→3)-β-D-glucan and subunit b has serine protease region. Factor G is supposed to be activated and express serine protease activity by the binding of (1→3)-β-D-glucan to subunit a. However, the complete structure of factor G remains unknown.

DISCLOSURE OF THE PRESENT INVENTION

The present invention was accomplished with these understandings and aims to isolate and purify the factor G derived from limulus amebocytes, particularly subunit a having a binding site with (1→3)-β-D-glucan and to elucidate its complete structure.

Another object of the present invention is to provide a polypeptide having a (1→3)-β-D-glucan binding site of a factor G and a DNA encoding thereof.

The other object of the present invention is to provide a polypeptide of subunit a of factor G and DNA encoding thereof.

Further object of the present invention is to provide a polypeptide of factor G containing subunit a and a DNA encoding thereof.

Therefore, the present invention relates to a single stranded DNA encoding for a polypeptide containing at least one motif structure shown by an amino acid sequence of (SEQ. ID No:34): Gln-Gln-Trp-Ser, a double stranded DNA composed of said DNA and its complementary DNA, and a polypeptide shown by said amino acid sequence.

Furthermore, the present invention relates to a single stranded DNA encoding for a polypeptide consisted of below mentioned amino acid sequence or a homologous sequence thereof, a double stranded DNA composed of said DNA and its complementary DNA and a polypeptide shown by said amino acid sequence (SEQ. ID No.2):

    __________________________________________________________________________     Ser                                                                               His                                                                               Glu                                                                               Pro                                                                               Lys                                                                               Trp                                                                               Gln                                                                               Leu                                                                               Val                                                                               Trp                                                                               Ser                                                                               Asp                                                                               Glu                                                                               Phe                                                                               Thr                                  Asn                                                                               Gly                                                                               Ile                                                                               Ser                                                                               Ser                                                                               Asp                                                                               Trp                                                                               Glu                                                                               Phe                                                                           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                                                                Gly                                                                               Phe                                                                               Lys                                                                               Tyr                                                                               Thr                                                                               Ser                                                                               Ala                                                                               Arg                                                                               Leu                                                                               Lys                                                                               Thr                                                                               Gln                                  Phe                                                                               Asp                                                                               Lys                                                                               Ser                                                                               Trp                                                                               Lys                                                                               Tyr                                                                               Gly                                                                               Lys                                                                               Ile                                                                               Glu                                                                               Ala                                                                               Lys            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                                                                     Asn                                                                               Trp                                                                               Ile                                                                               Arg                                                                               Ile                                                                               Thr                                                                               Lys                                                                               Val                                                    __________________________________________________________________________

Four species of horseshoe crab have been known and the coagulogens which are components of amebocytes of these four species of horseshoe crab are very similar in their structure (73%-9%) that is necessary for the expression of their function. Thus, the polypeptides of the present invention derived from these four species of horseshoe crab are presumed to have similar amino acid sequence as well as that in coagulogen.

In other words, where there is a homology between polypeptides, there is an identical or similar functional expression with their similar amino acid sequences though they have no identical amino acid sequences. Therefore, person skilled in the art will easily understand that the present invention encompasses not only the above mentioned amino acid sequence but also its homologous amino acid sequences.

The present invention will be explained in detail.

Factor G can be isolated and purified from amebocyte lysate of horseshoe crab by various column chromatography techniques, for example, affinity chromatography with dextran sulfate-Sepharose^(RTM) CL-6B and Concanavalin A-Sepharose^(RTM) 4B, gel filtration chromatography such as Sephacryl^(RTM) S-200HR (T. Morita et al. (1981) FEBS Lett., 129(2), 318-321, Japanese Published Examined Patent Application (herein after abbreviated as Japan Tokkyo Koho) No. 399 (1991). Furthermore, subunits a and b which constitute factor G can be obtained by denaturation of factor G with a denaturing agent such as a surfactant followed by fractionation with high performance liquid chromatography (hereinafter abbreviated as HPLC) using a molecular sieve. The partial amino acid sequences of the each subunits can be determined by the following; each subunit is subjected to reduction and alkylation and enzymic digestion to give peptide fragments, and the amino acid sequence of the peptide fragments is determined by a peptide sequencer and so on. cDNAs encoding for the each subunits are isolated from the cDNA libraries, which are prepared from poly(A)⁺ RNA isolated from limulus amebocytes, using antibodies to each subunits or oligonucleotides synthesized according to aforementioned partial amino acid sequences of each subunits. Then the nucleotide sequence of these cDNA can be determined using dideoxy chain termination method (Sanger, F. et al., Proc. Natl. Acad. Sci. U.S.A., 74, 5463-5467 (1977)). The amino acid sequences of each subunits can be determined from above mentioned nucleotide sequences and partial amino acid sequences.

cDNA (AGC . . . GTG; base Nos. 114-2,075) encoding for polypeptide of subunit a of factor G shown in sequence No. 1 and the corresponding amino acid sequence (Ser . . . Val; amino acid Nos. 1-654) are determined according to the above mentioned method.

Further, subunit a of factor G was found to have a domain structure from its amino acid sequence.

That is, a glucanase domain having amino acid sequence similar to that of carboxyl terminal of β-1,3-glucanase was found in the amino terminal of subunit a (Pro . . . Ala; amino acid Nos. 4-236) (FIG. 2). While, the carboxyl terminal portion of subunit a has a repetitive structure composed of 126 amino acids (Ser . . . Ile; amino acid Nos. 391-516 and Ser . . . Val; amino acid Nos. 529-654), and this sequence is similar to that of amino terminal portion of xylanase Z (FIG. 3). Furthermore, a "QQWS (Gln-Gln-Trp-Ser)" SEQ. ID No.34motif is contained between domains of glucanase and xylanase, and three repetitions of a sequence similar to that of xylanase A (Leu . . . Leu; amino acid Nos. 247-293, Glu . . . Val; amino acid Nos. 294-340 and Gly . . . Ser; amino acid Nos. 341-387) (FIG. 4).

Additionally, FIG. 2-4 showed each amino acid with one letter code.

In the present invention, recombinant DNA vectors including the DNA encoding for these peptides are prepared and transfected into hosts and cultured or bred with so-called gene technology to produce and collect desired peptides.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 shows the mechanisms of endotoxin sensitive and (1→3)-β-D-glucan sensitive coagulation cascade reaction of horseshoe crab amebocyte.

FIG. 2 shows the amino acid sequence of glucanase domain of subunit a (amino acid Nos. 4-236). In the Fig., FGA indicates an amino acid sequence of amino acid Nos. 4-236 of subunit a of factor G, and : GI Al indicates an amino acid sequence of amino acid Nos. 421-682 of β-1,3-glucanase.

FIG. 3 shows a domain of amino acid Nos. 391-654 in subunit a of factor G. In the Fig., FGA indicates an amino acid sequence of amino acid Nos. 391-654 of subunit a of factor G, and Xyn Z indicates an amino acid sequence of amino acid Nos. 298-434 of xylanase Z.

FIG. 4 shows a domain of amino acid Nos. 247-387 of subunit a of factor G. In the Fig., FGA indicates an amino acid sequence of amino acid Nos. 247-387 of subunit a of factor G, and Xln A indicates an amino acid sequence of amino acid Nos. 351-477 of xylanase A.

THE BEST MODE TO PERFORM THE INVENTION

The present invention will be explained by the following practical examples, however, the scope of the invention is not restricted by these examples.

EXAMPLE 1 1. Purification of Factor G

(1) Preparation of amebocyte lysate 400 ml of 0.02M Tris-HCI buffer containing 50 mM NaCl, pH 8.0 was added to 113 g of amebocytes of Tachypleus tridentatus, and homogenized for three minutes with a high speed homogenizer (Hyscotron^(RTM), Nippon Seimitsu Kogyo Co., Ltd.) and then centrifuged at 8,000 rpm for 30 min. to obtain a supernatant. Further, 300 ml of the same buffer was added to the resultant precipitates, homogenized and centrifuged in a similar manners to obtain a supernatant. The similar procedure was repeated further twice. All supernatants obtained by centrifugation were combined and 1,250 ml of amebocyte lysate was obtained.

(2) Purification of factor G from the amebocyte lysate.

a. Dextran sulfate-Sepharose^(RTM) CL-6B column chromatography.

The obtained 1,250 ml of extract was applied to a dextran sulfate-Sepharose^(RTM) CL-6B column (5×17.8 cm) equilibrated in advance with the extraction buffer. The preparation of the column is performed according to the method shown in Japan Tokkyo koho No. 399 (1991) preparative example 2. The applied column was washed thoroughly with the same buffer and washed with 0.02M Tris-HCl buffer containing 0.15M NaCl, pH 8.0, to wash out the excess amount of protein which adsorbed into the column. The adsorbed protein into the column was eluted with 0.02M Tris-HCl buffer containing 0.25M NaCi, pH 8.0, to obtain factor G fraction.

b. Concanavalin A-Sepharose^(RTM) 4B column chromatography.

The above mentioned factor G fraction was applied to a Con A-Sepharose^(RTM) 4B column (Pharmacia Biotech K.K.) (2×16 cm) equilibrated with 0.02M Tris-HCl buffer containing 0.25M NaCi, pH 8.0, washed thoroughly with the equilibration buffer and 0.02M Tris-HCl buffer containing 0.5M NaCl, pH 8.0, successively, then, the protein which adsorbed into the column was eluted with 0.02M Tris-HCl buffer containing 0.5M each of NaCl and methyl α-D-glucoside, pH 8.0, to obtain factor G fraction.

c. Sephacryl^(RTM) S-200 HR column chromatography

The above mentioned factor G fraction was concentrated with ultrafiltration and applied to a Sephacryl^(RTM) S-200 HR column (Pharmacia Biotech K.K.) (2.7×98 cm) equilibrated with 0.05M sodium phosphate buffer, pH 6.5, and protein was eluted with the same buffer. The final eluate fraction of this chromatography containing the protein with slight absorbance at 280 nm was collected to obtain the protein of finally purified preparation of factor G. About 300 μg of factor G calculated as bovine serum albumin was obtained from 113 g of amebocytes.

2. Determination of Partial Ssequence of Subunit a of Factor G

(1) Separation of subunits a and b of factor G

Subunits a and b of factor G were separated for the elucidation of partial amino acid sequences of subunits a and b required for cDNA cloning of subunits a and b of factor G. The factor G obtained in the above mentioned 1 was precipitated with methanol and chloroform for desalting and concentration (Methods in Enzymology, 182, p. 78-79 (1990)). The resultant precipitates were dissolved in 2% sodium dodecylsulfate (SDS) solution, and heated at 100° C. for 5 min. This heated sample was applied to gel filtration by using a tandem columns of TSKgeI^(RTM) G3000SW (TOSOH Corp.) using 0.1M sodium phosphate buffer containing 0.1% SDS, pH 7.0, as a mobile phase, and subunits a and b of factor G were separated.

(2) Enzymic digestion of subunit a of factor G

Trichloroacetic acid (TCA) was added to give final concentration of 17.4 w/v % into the fraction of subunit a of factor G which obtained by above mentioned 2(1), and subunit a of factor G was precipitated. According to the method of Paul Matsudaira (A Practical Guide to Protein and Peptide Purification for Microsequencing, p. 42-43, 1989, Academic Press, Inc.), the precipitated subunit a of factor G was dissolved in 0.4M ammonium bicarbonate containing 8M urea and caused to reduction and alkylation with iodoacetamide. The reaction mixture was added with water to give 4M urea concentration, and lysyl endopeptidase (Wako Pure Chemical Ind. Ltd.) was added at an enzyme-suibstrate weight ratio of 1:60 and digestion was performed at 37° C. for 20 hrs. The digested product was applied to a μBondasphere^(RTM) 5μC8-300 angstrom (Waters Co., Ltd.) column (2.1×150 mm) preliminarily equilibrated with 0.06 v/v % trifluoroacetic acid and the column was thoroughly washed and eluted with acetonitrile using a linear concentration gradient method of 0 v/v % at 5 min., 24 v/v % at 65 min., 56 v/v % at 125 min. and 80 v/v % at 135 min. at a flow rate of 0.2 ml/min. to elute the adsorbed protein. The eluted peptides were monitored with UV absorbance at 210 nm, and completely collected.

(3) Determination of partial amino acid sequence.

The amino acid sequence of obtained each peptide was determined with a gas phase sequencer Model 477A (Applied Biosystems Japan Inc.). The results are shown in Table 1.

                                      TABLE 1                                      __________________________________________________________________________     Peptide 1 (SEQ. ID NO.5)                                                       SerXaaGluProLysXaaGlnLeuVal                                                    Peptide 2 (SEQ. ID NO.6)                                                       ArgGluAspTyrAspGlyPheLys                                                       Peptide 3 (SEQ. ID NO.7)                                                       TyrThrSerAlaArgLeuLys                                                          Peptide 4 (SEQ. ID NO.8)                                                       ThrGlnPheAspLys                                                                Peptide 5                                                                      SerTrpLys                                                                      Peptide 6                                                                      TyrGlyLys                                                                      Peptide 7 (SEQ. ID NO.9)                                                       MetAlaIleProSerPheArgGlyValTrpValMetPhe                                        TrpMetSerGlyXaaAsnThrAsnTyrValXaaXaaPro                                        Peptide 8 (SEQ. ID NO.10)                                                      SerAlaPheArgAsnLys                                                             Peptide 9 (SEQ. ID NO.11)                                                      ValPheValIleLeuAsnMetAlaIleGlyGlyAsnXaa                                        ProGlyPheXaaValAla                                                             Peptide 10 (SEQ. ID NO.12)                                                     IleIleProArgGlySerGlyLys                                                       Peptide 11 (SEQ. ID NO.13)                                                     ValGlnLeuGluAspThrSerAspValGlyGlyGlyLys                                        Peptide 12 (SEQ. ID NO.14)                                                     CysAspAsnGluGlyAlaTrpMetAlaTyrLys                                              Peptide 13 (SEQ. ID NO.15)                                                     AspIleAspPheProSerSerGlyAsnTyrXaaIleGlu                                        TyrXaaValAla                                                                   Peptide 14 (SEQ. ID NO.16)                                                     LeuIleGlnAlaGluXaaTyrPheXaaTyrXaaGluVal                                        GlnLeuGlu                                                                      Peptide 15 (SEQ. ID NO.17)                                                     GluGlyAlaTrpMetAlaTyrLys                                                       Peptide 16 (SEQ. ID NO.18)                                                     ValArgGlyThrIleHisTrpSerThrProAspGlyAla                                        HisAlaHisHisAsnArg                                                             __________________________________________________________________________

In the formula, Xaa represents one of naturally occurring amino acids.

3. Synthesis of Oligonucleotide

In the determined amino acid sequences shown in Table 1, two amino acid sequences A (SEQ. ID No.6): -Glu-Asp-Tyr-Asp-Gly-Phe- and B(SEQ. ID No.9): -Trp-Val-Met-Phe-Trp-Met- were selected and reverse translated as a sense in the case of A and as an antisense in the case of B. A mixture of 25 mer and 26 mer oligonucleotides shown below having recognition sequence of restriction enzyme and two bases for protection of DNA at 5'-terminal were synthesized with a DNA synthesizer 380A (Applied Biosystems Japan Inc.). ##STR1##

The oligonucleotides A' and B' shown above include all possibilities of sequences corresponding to A and B or complementary sequences thereof (however, the third nucleotide (T, C) in codon of Phe in A (TT(C/T)) was excluded).

4. Preparation of PolY(A)⁺ RNA Containing mRNA Encoding for Factor G

Since poly(A)⁺ RNA was isolated from amebocytes of horseshoe crab, factor G is purified from amebocytes of horseshoe crab.

(1) Preparation of total RNA

By using of AGPC method (Experimental Medicine (Jikken Igaku) 9, 1937-1940 (1991), Pub. by Yodosha Co., Ltd.), about 11 mg of total RNA was isolated from 11.8 g of limulus amebocytes.

(2) Preparation of poly(A)⁺ RNA Poly(A)⁺ RNA was isolated from about 2 mg of the above mentioned total RNA with Oligotex-dT^(RTM) 30 Super kit (Nippon Roche K.K.). The similar procedure was repeated once again for further purification to obtain 34.5 μg of highly purified poly(A)⁺ RNA from 2 mg of total RNA.

5. Preparation of cDNA Library of Amebocytes of Horseshoe crab

(1) Synthesis of cDNA

cDNA was synthesized from 5 μg in 34.5 μg of poly(A)⁺ RNA obtained in the above mentioned 4 with a cDNA synthesis kit (Amersham Co. Ltd.).

(2) Preparation of cDNA library

cDNA library of amebocytes of horseshoe crab was prepared from cDNA synthesized in the above mentioned (1) by a cDNA cloning system λgt10 adapter method (Amersham Co. Ltd.).

6. cDNA cloning of subunit a of factor G

cDNA fragment encoding for a part of subunit a of factor G was amplified with the oligonucleotide prepared in the above mentioned 3 using poly(A)⁺ RNA prepared in the above mentioned 4 (2) as a template by PCR method (Saiki, R. K., et al. Science, 239, 487-491, (1988)). The amplified cDNA fragment was labeled with α-³² P!dCTP using Multiprime-DNA labeling kit (Nippon Gene Co., Ltd.) to obtain a probe. The probe was used for the screening of cDNA library prepared in above mentioned 5 (2) to obtain two positive clones containing a longest insert cDNA having about 2,400 bp. The insert cDNA of these clones were almost identical except for the length difference of 5'- and 3'-terminals in several bases, and their nucleotide sequences were determined. The composite nucleotide sequence contained an initiation codon and poly(A)⁺ tail, and showed the length of 2,408 bp.

7. Determination of Nucleotide Sequence of cDNA Encoding for Subunit a of Factor G

The insert cDNA prepared in the above mentioned 6 was integrated in pUC118 vector and pBluescript Π SK vector. The determination of total nucleotide sequences of cDNA cloned in pUC118 vector and pBluescript Π SK vector was performed by subcloning with deletion using restriction enzyme recognition site on cDNA fragment and kilo-sequence deletion kit (Takara Shuzo Co., Ltd.). The nucleotide sequence of cDNA in the clone prepared by the above mentioned procedure was determined with a DNA sequencer 370A (Applied Biosystems Japan Inc.) using fluorolabeled nucleotide primer (Smith, L. M. et al. (1986), Nature 321, 674-679).

The determined nucleotide sequence of cDNA of subunit a of factor G, and the deduced amino acid sequence were shown in SQ ID No. 1 of the Sequence listing. The partial amino acid sequences determined in 2 (3) (Table 1) are all included in the above mentioned amino acid sequence, and the inserted cDNA whose nucleotide sequence was determined by the procedure, was confirmed its encoding of subunit a of factor G.

8. Expression and Purification of Factor G or Subunits Thereof

Whole or partial gene encoding for subunit a of factor G was cut out from the clone obtained above with ultrasonic wave or restriction enzyme treatment, or the other methods known in the art and integrated to a suitable vector. Vectors which constructed from such as phages or plasmids which autonomously proliferate in host microorganisms or cells for gene recombination including suitable promoters, SD sequences, translation initiating codon ATG, and suitable structural gene can be preferably used. These constructed vectors are integrated to obtain transformants in a suitable host organisms or cells, such as various strains of E. coli and yeasts, animal cells (e.g. oocytes of mice and rats, Chinese hamster oocytes (CHO)), plant cells and insect cells or animal, plant or insect hosts. The resultant transformant is cultured in a nutrient medium or bred with nutrient feeds to have stable production of large amount of factor G or subunit thereof (hereinafter abbreviated as factor G etc.). Culture or breeding conditions of the transformant may be suitably modified within the scope of factor G etc. production and conventional conditions known well to persons skilled in the art for the growth of hosts can be preferably used. Factor G etc. in the cultured products or bred animals can be collected from extracts of mechanically ground cultured solution containing microorganisms or cells, or individuals. However, when factor G etc. exist in the cultured solutions or extracts, factor G etc. are generally used after separating solutions containing factor G etc. from microorganisms, cells or ground individuals by filtration or centrifugation. When factor G etc. exist in microorganisms, cells, or organ membranes of individuals, microorganisms, cells or particles are collected by the filtration or centrifugation of the obtained cultured products or particles, and solubilized by mechanical or ultrasonic treatment, enzymic treatment with lysozyme and so on, addition of a chelating agent such as EDTA and/or a surfactant to separate and collect the factor G etc. The solutions containing factor G etc. obtained above can be isolated by conventional protein purification methods.

Factor G etc. expressed as a chimera polypeptide can be easily isolated by application of the properties of the other proteins. For example, the aimed factor G etc. can be easily isolated with affinity chromatography by applying the affinity of the other proteins.

Practically, subunit a of factor G can be expressed by the following procedure.

An oligonucleotide having a sequence of (SEQ. ID No.21)5'-AGCCACGAACCAAAGTGGCA-3' is synthesized and its 5'-terminal is phosphorylated. The resultant oligonucleotide is annealed with a single-stranded DNA prepared from a plasmid integrated with a gene encoding for the subunit a of factor G, and elongation reaction was performed with DNA polymerase such as Klenow fragment. The single-stranded portion produced in this procedure is digested with a DNA nuclease such as Mung bean nuclease to obtain a double-stranded DNA. The resultant double-stranded DNA is digested with Sph I restriction enzyme and the produced cohesive ends are blunted with T4 DNA polymerase, Klenow fragment or Blunting kit. This double-stranded DNA is preliminarily digested with Nco I restriction enzyme and then the resultant cohesive end is integrated to pTV118N contained ampicillin resistant gene which was blunted with T4 DNA polymerase, Klenow fragment or a Blunting kit. The produced plasmid is transfected into E. coli JM109 or E. coli MV1184 to obtain transformant. The aimed transformant can be selected by applying conventional methods using the properties of plasmid. That is, a transformed microorganism is cultured on a LB plate contained ampicillin which is preliminarily sprayed with 5-bromo-4-chloro-3-(3-indolyl)-β-D-galactoside (X-gal)/isopropylthiogalactoside (IPTG) and white colonies which transducted with the aimed plasmid are picked up and selected. Further screening with a synthetic oligonucleotide probe of (SEQ. ID No.22)5'-AAACAGACCATGAGCCACGAACCA-3' gave the plasmid with correct sequence. Furthermore, it can be confirmed by DNA sequencing with a sequencing primer using RV-N primer (Takara Shuzo Co., Ltd.) whether the plasmid was constructed correctly. E. coli JM109 or E. coli MV1184 transfected with the plasmid with correct sequence is cultured in a 3% Nutrient broth (Nissui Pharmaceutical Co., Ltd.) containing 100 μg/ml ampicillin and 1 mM IPTG at 30° C. for 24 hrs. Subunit a of factor G is purified the cultured supernatant or extract of microorganisms by conventional methods. An affinity chromatography with antibody or a ligand having affinity to subunit a of factor G is preferably used for the more simple purification.

EXAMPLE 2 1. Determination of Partial Amino Acid Sequence of Subunit b of Factor G

(1) Isolation of subunit b of factor G

Subunit b can be similarly isolated according to Example 1, 2. (1) by separation of subunits a and b.

(2) Enzymic digestion of subunit b of factor G

Peptide fragments were obtained by the same method as Example 1, 2. (2) from subunit b of factor G obtained by above mentioned (1).

(3) Determination of partial amino acid sequence

The respective amino acid sequence of obtained peptides were determined by a similar manner to that of Example 1, 2. (3). The results are shown in Table 2.

                                      TABLE 2                                      __________________________________________________________________________     Peptide 1 (SEQ. ID NO.23)                                                      GlyIleAsnGluLys                                                                Peptide 2 (SEQ. ID NO.24)                                                      XaaXaaGlyPheXaaProValIleThr                                                    Peptide 3 (SEQ. ID NO.25)                                                      IleIleGlyGlyGlyIleAlaThrProHisSerXaaPro                                        XaaMetValGlyIlePhe                                                             Peptide 4                                                                      ValAsnPro                                                                      Peptide 5 (SEQ. ID NO.26)                                                      ValXaaValValThrAlaAlaHisCysLeuValThrGln                                        PheGlyAsnArgGlnXaaTyrSerIlePheValArgVal                                        GlyAlaHisXaaIleXaaAsnSerGlyThrAsn                                              Peptide 6 (SEQ. ID NO.27)                                                      ValValIleThrGlyTrpGlyValThrGlyLys                                              Peptide 7 (SEQ. ID NO.28)                                                      AsnValLeuArgGluLeuGluLeuProValValThrAsn                                        GluGlnCysXaaLys                                                                Peptide 8 (SEQ. ID NO.29)                                                      SerTyrGlnThrLeuProPheSerLys                                                    Peptide 9 (SEQ. ID NO.30)                                                      LeuAsnArgGlyIleThrAsnAspMetIleCysAlaGly                                        PheProGluGlyGlyLys                                                             Peptide 10 (SEQ. ID NO.31)                                                     AspAlaCysGlnGlyAspSerGlyGlyProLeuMetTyr                                        GlnAsnProThrThrGlyArgValLys                                                    __________________________________________________________________________

2. Synthesis of Oligonucleotide

In the determined amino acid sequences, two amino acid sequences C: -Asn-Glu-Gln-Cys-(Asn)-Lys and D: -Met-Tyr-Gln-Asn-Pro-Thr- were used for reverse translation of C and D as sense and antisense, respectively, and the mixture of oligonucleotides C' and D' each having 25 nucleotides as shown below were synthesized in a similar manner to that of Example 1, 3. In the C, -(Asn)- means presumed as Asn. ##STR2##

The oligonucleotides C' and D' shown above include possibility of all the sequences corresponding to C and D, or complementary sequences. However, the C-terminal amino acid Lys of C and D, and Thr in D exclude the third nucleotides (A and G, and T, C, A, and G, respectively) in the respective codon of AA(A/G) and AC(A/C/G/T).

4. cDNA Cloning of Subunit b of Factor G

Using the poly(A)⁺ RNA obtained by Example 1, 4. (2) as a template and the oligonucleotide synthesized by the above mentioned 2, the experiment was performed in a similar manner to that of Example 1, 6., and a positive clone having 1,979 bp of cDNA was obtained. The nucleotide sequence of insertion cDNA in the clone was analyzed and included a nucleotide sequence corresponding to amino acid sequence of peptide derived from subunit b of factor G obtained by the above mentioned 1. (2), and poly A additional signal. The clone was presumed to have full length of cDNA from its size.

5. Determination of cDNA Nucleotide Sequence Encoding for Subunit b of Factor G

The nucleotide sequence of cDNA of subunit b of factor G was determined by a similar manner to that of Example 1, 7. The determined nucleotide sequence of cDNA of subunit b of factor G and amino acid sequence determined from the nucleotide sequence is shown in sequence No. 2 in the Sequence Listing.

The amino acid sequence includes all of partial amino acid sequences determined by Example 2, 1 (3) and shown in Table 2, thus, the determined nucleotide sequence of insertion cDNA surely encoding for subunit b of factor G.

The recombinant DNA vector containing cDNA encoding for the polypeptide obtained by Example 2 was transfected into reproducible host microorganisms, suitable animal cells, insect or cells thereof. They were screened using a marker of vector and (1→3)-β-D-glucan binding ability as indicators. The polypeptide of the present invention can be obtained by culturing or breeding of microorganisms, animal cells, insect or cells thereof having the recombinant DNA vector.

Industrial Applicability

In the present invention, subunit a of factor G of amebocyte lysate of horseshoe crab was isolated and purified, and the amino acid sequence and cDNA nucleotide sequence encoding for said amino acid sequence were determined.

The resultant sequence included a sequence of (1→3)-β-D-glucan binding region, and polypeptide corresponding to the binding region can be obtained by gene technological methods using the cDNA. The obtained polypeptide exhibits the following various effects.

(1) Recent increase of immunodisorder patients and aged populations markedly increased patients with opportunistic infections with secondary pathogens such as Candida and Aspergillus which generally have weak pathogenicity. However, their clinical diagnosis, particularly deep-seated mycoses of organs, was difficult without invasive procedure except special patterns of diseases and often definitely diagnosed by post-autopsy (Encyclopedia of Microbiology, Gihodo Shuppan Co., Ltd., 1989).

The diagnosis of deep-seated mycoses can be carried out by measuring (1→3)-β-D-glucan with ligand-receptor assay method using labeled polypeptide contained the (1→3)-β-D-glucan binding region or (1→3)-β-D-glucan labeled with radioactive isotopes, enzymes, fluorescent or luminous compounds. Said polypeptide may be immobilized on a solid phase such as microplate, test tube or beads for the specific detection of (1→3)-β-D-glucan. These measuring methods specifically measure (1→3)-β-D-glucan derived from fungi in the body fluid for simple and rapid diagnosis of deep-seated mycoses.

(2) Medicines composed of an antifungal agent - conjugated polypeptide having a binding region with (1→3)-β-D-glucan exhibit potent affinity with lesions, and it will be new selective antifungal agents which specifically kill fungi which have (1→3)-β-D-glucan on their cell walls exist in lesion.

(3) Furthermore, recent advances of gene manipulation technology often use yeasts as hosts of vector containing gene encoding for the aimed protein in its expression. This is because the production of glycoprotein is possible and mass culture is possible as well as bacteria. However, very small amount of residues of yeast having as a component often accompanies in the products, and its measurement and removal are required. An affinity chromatography with an affinity carrier of polypeptide contained (1→3)-β-D-glucan binding region bound to support may specifically remove or determine the residue of yeasts. In addition, confirmation of the absence of yeast cell component in the product after removal was performed by the measurement using polypeptide contained (1→3)-β-D-glucan binding site as described in (1) and provided more simple and specific confirmation than that of limulus amebocyte lysate.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 39                                                  (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2409 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 114..2075                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        GTGGGGGGTTTAGTTGAAACAGTAAATACGTTAACTGTTTAATCTTGTTAATTGCAATGT60                 TGGTGTTGCTGTGTTGTGTTGTTTTGCATGTTGGTGTTGCAAGAATTTGCTGTAGC116                    Ser                                                                            CACGAACCAAAGTGGCAGCTCGTCTGGTCGGATGAATTTACCAATGGA164                            HisGluProLysTrpGlnLeuValTrpSerAspGluPheThrAsnGly                               51015                                                                          ATAAGTTCTGATTGGGAATTTGAAATGGGCAATGGCCTCAATGGTTGG212                            IleSerSerAspTrpGluPheGluMetGlyAsnGlyLeuAsnGlyTrp                               202530                                                                         GGTAATAACGAACTGCAATATTATCGTCGTGAAAATGCCCAAGTTGAG260                            GlyAsnAsnGluLeuGlnTyrTyrArgArgGluAsnAlaGlnValGlu                               354045                                                                         GGAGGGAAACTGGTAATTACTGCTAAAAGAGAAGACTATGATGGCTTC308                            GlyGlyLysLeuValIleThrAlaLysArgGluAspTyrAspGlyPhe                               50556065                                                                       AAATACACTTCTGCTAGGCTGAAAACCCAGTTTGATAAATCTTGGAAG356                            LysTyrThrSerAlaArgLeuLysThrGlnPheAspLysSerTrpLys                               707580                                                                         TATGGTAAAATTGAAGCCAAAATGGCGATTCCATCATTTCGGGGAGTC404                            TyrGlyLysIleGluAlaLysMetAlaIleProSerPheArgGlyVal                               859095                                                                         TGGGTGATGTTCTGGATGTCAGGAGACAACACTAATTATGTTAGATGG452                            TrpValMetPheTrpMetSerGlyAspAsnThrAsnTyrValArgTrp                               100105110                                                                      CCATCTTCTGGTGAAATTGACTTTATTGAACATAGAAACACTAACAAT500                            ProSerSerGlyGluIleAspPheIleGluHisArgAsnThrAsnAsn                               115120125                                                                      GAAAAAGTCAGAGGAACTATTCACTGGTCCACTCCTGACGGTGCTCAT548                            GluLysValArgGlyThrIleHisTrpSerThrProAspGlyAlaHis                               130135140145                                                                   GCGCATCATAACAGAGAAAGTAATACAAATGGGATTGATTATCACATT596                            AlaHisHisAsnArgGluSerAsnThrAsnGlyIleAspTyrHisIle                               150155160                                                                      TATTCTGTAGAGTGGAATTCTTCCATTGTTAAATGGTTTGTTAATGGA644                            TyrSerValGluTrpAsnSerSerIleValLysTrpPheValAsnGly                               165170175                                                                      AATCAATACTTTGAAGTGAAAATTCAGGGAGGAGTAAATGGGAAAAGT692                            AsnGlnTyrPheGluValLysIleGlnGlyGlyValAsnGlyLysSer                               180185190                                                                      GCATTTCGTAACAAAGTTTTCGTTATTTTAAACATGGCGATTGGTGGA740                            AlaPheArgAsnLysValPheValIleLeuAsnMetAlaIleGlyGly                               195200205                                                                      AACTGGCCAGGATTCGATGTTGCTGACGAGGCTTTCCCTGCTAAAATG788                            AsnTrpProGlyPheAspValAlaAspGluAlaPheProAlaLysMet                               210215220225                                                                   TACATTGATTATGTCCGTGTATACCAGGATGCCAGTACATCTTCTCCT836                            TyrIleAspTyrValArgValTyrGlnAspAlaSerThrSerSerPro                               230235240                                                                      GTTGGGGATACCTCTTTAGATGGTTACTATTTTGTCCAAAACAGGCAC884                            ValGlyAspThrSerLeuAspGlyTyrTyrPheValGlnAsnArgHis                               245250255                                                                      AGTGAATTGTATCTTGATGTCACTGATGCCAGTAACGAAGATGGAGCA932                            SerGluLeuTyrLeuAspValThrAspAlaSerAsnGluAspGlyAla                               260265270                                                                      TTTCTGCAACAATGGTCTTATAGTGGTAATGAGAACCAACAGTTTGAT980                            PheLeuGlnGlnTrpSerTyrSerGlyAsnGluAsnGlnGlnPheAsp                               275280285                                                                      TTTGAGCATCTCGAAAATAATGTTTATAAAATTACTAATAAAAAAAGT1028                           PheGluHisLeuGluAsnAsnValTyrLysIleThrAsnLysLysSer                               290295300305                                                                   GGAAAATCTTTGGATGTTTATAATTTTGGGACTGAGAATGGTGTTAGA1076                           GlyLysSerLeuAspValTyrAsnPheGlyThrGluAsnGlyValArg                               310315320                                                                      ATCCAACAGTGGTCATATGGAGGGGCTCGCAATCAGCAGTTTACTGTA1124                           IleGlnGlnTrpSerTyrGlyGlyAlaArgAsnGlnGlnPheThrVal                               325330335                                                                      CAAAGTGTTGGTGATGGTTATTATAAGATTATTCCACGCGGCAGTGGA1172                           GlnSerValGlyAspGlyTyrTyrLysIleIleProArgGlySerGly                               340345350                                                                      AAGTTAGTGGAAGTAGCAGATTTTAGTAAAGATGCAGGAGGGAAGATA1220                           LysLeuValGluValAlaAspPheSerLysAspAlaGlyGlyLysIle                               355360365                                                                      CAACAATGGTCTGATAACAACCAATTATCTGGACAGTGGAAACTTATT1268                           GlnGlnTrpSerAspAsnAsnGlnLeuSerGlyGlnTrpLysLeuIle                               370375380385                                                                   AAAAGTAAAAGTTATTCTAAATTAATTCAGGCAGAAAGTTATTTTGAT1316                           LysSerLysSerTyrSerLysLeuIleGlnAlaGluSerTyrPheAsp                               390395400                                                                      TCCTCAAAAGTACAATTGGAAGATACCTCAGATGTAGGAGGTGGGAAG1364                           SerSerLysValGlnLeuGluAspThrSerAspValGlyGlyGlyLys                               405410415                                                                      AATGTTAAATGTGATAATGAAGGAGCCTGGATGGCTTATAAGGATATT1412                           AsnValLysCysAspAsnGluGlyAlaTrpMetAlaTyrLysAspIle                               420425430                                                                      GATTTCCCCAGTTCAGGTAATTATCGAATAGAATACAGAGTAGCAAGT1460                           AspPheProSerSerGlyAsnTyrArgIleGluTyrArgValAlaSer                               435440445                                                                      GAACGTGCAGGAGGAAAGCTGTCTCTGGATTTGAATGCAGGCTCTATA1508                           GluArgAlaGlyGlyLysLeuSerLeuAspLeuAsnAlaGlySerIle                               450455460465                                                                   GTTCTTGGCATGCTGGATGTTCCTTCAACAGGAGGATGGCAGAAGTGG1556                           ValLeuGlyMetLeuAspValProSerThrGlyGlyTrpGlnLysTrp                               470475480                                                                      ACCACCATTTCCCATACAGTGAATGTGGATTCAGGTACATATAACTTG1604                           ThrThrIleSerHisThrValAsnValAspSerGlyThrTyrAsnLeu                               485490495                                                                      GGGATCTATGTTCAACGAGCCAGCTGGAATATCAACTGGATAAAGATT1652                           GlyIleTyrValGlnArgAlaSerTrpAsnIleAsnTrpIleLysIle                               500505510                                                                      ACAAAAATACCTGAACAGTCAAATTTGAATCAAGGGCGTCGTAATTCT1700                           ThrLysIleProGluGlnSerAsnLeuAsnGlnGlyArgArgAsnSer                               515520525                                                                      AAATTAATTCAGGCAGAAAGTTATTTTAGTTACTCAGAAGTACAACTG1748                           LysLeuIleGlnAlaGluSerTyrPheSerTyrSerGluValGlnLeu                               530535540545                                                                   GAAGATACCTTAGATGTAGGAGGTGGAAAGAATGTTAAATGTGATAAA1796                           GluAspThrLeuAspValGlyGlyGlyLysAsnValLysCysAspLys                               550555560                                                                      GAAGGGGCCTGGATGGCTTACAAGGATATTGATTTCCCCAGTTCAGGA1844                           GluGlyAlaTrpMetAlaTyrLysAspIleAspPheProSerSerGly                               565570575                                                                      AGTTATCGAGTAGAATACAGAGTGGCAAGTGAACGTGCAGGAGGAAAG1892                           SerTyrArgValGluTyrArgValAlaSerGluArgAlaGlyGlyLys                               580585590                                                                      CTGTCCCTAGATTTGAATGCAGGCTCTATAGTGCTTGGCATGCTGGAT1940                           LeuSerLeuAspLeuAsnAlaGlySerIleValLeuGlyMetLeuAsp                               595600605                                                                      ATTCCTTCAACAGGAGGATTGCAGAAGTGGACCACCATTTCTCATATA1988                           IleProSerThrGlyGlyLeuGlnLysTrpThrThrIleSerHisIle                               610615620625                                                                   GTGAATGTGGATTTAGGTACATATAACTTGGGAATTTATGTTCAAAAA2036                           ValAsnValAspLeuGlyThrTyrAsnLeuGlyIleTyrValGlnLys                               630635640                                                                      GCCAGTTGGAATATCAATTGGATTAGAATTACAAAAGTGTAGGATACAA2085                          AlaSerTrpAsnIleAsnTrpIleArgIleThrLysVal                                        645650                                                                         GAGCAAACCAATTGTATTATTTTGAAGAAACAACAGCTGTTGACCATAATCTTTGTTCAT2145               TGAGAATTTATCCAACTGTTATAGAATCTATCACCTTTCCAGATGTAACGCATTGCTGAT2205               GGTTTTGAACTAATAAATGAGGAGATTATAAGTGCTAATGTGTTTGTTATATCTTTAATT2265               TTTAAAAACAAATTATCAACTAACTTTTCAATTCAGGCATGGTGTTTCTCTTTTTAATCT2325               GTATTTCTAATAAATTAATGTCTTTAAGAGTTGTTTTGTTTACAATAAATAAAGTTTGAT2385               TGTGTGGGATAAAAAAAAAAAAAA2409                                                   (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 654 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        SerHisGluProLysTrpGlnLeuValTrpSerAspGluPheThrAsn                               151015                                                                         GlyIleSerSerAspTrpGluPheGluMetGlyAsnGlyLeuAsnGly                               202530                                                                         TrpGlyAsnAsnGluLeuGlnTyrTyrArgArgGluAsnAlaGlnVal                               354045                                                                         GluGlyGlyLysLeuValIleThrAlaLysArgGluAspTyrAspGly                               505560                                                                         PheLysTyrThrSerAlaArgLeuLysThrGlnPheAspLysSerTrp                               65707580                                                                       LysTyrGlyLysIleGluAlaLysMetAlaIleProSerPheArgGly                               859095                                                                         ValTrpValMetPheTrpMetSerGlyAspAsnThrAsnTyrValArg                               100105110                                                                      TrpProSerSerGlyGluIleAspPheIleGluHisArgAsnThrAsn                               115120125                                                                      AsnGluLysValArgGlyThrIleHisTrpSerThrProAspGlyAla                               130135140                                                                      HisAlaHisHisAsnArgGluSerAsnThrAsnGlyIleAspTyrHis                               145150155160                                                                   IleTyrSerValGluTrpAsnSerSerIleValLysTrpPheValAsn                               165170175                                                                      GlyAsnGlnTyrPheGluValLysIleGlnGlyGlyValAsnGlyLys                               180185190                                                                      SerAlaPheArgAsnLysValPheValIleLeuAsnMetAlaIleGly                               195200205                                                                      GlyAsnTrpProGlyPheAspValAlaAspGluAlaPheProAlaLys                               210215220                                                                      MetTyrIleAspTyrValArgValTyrGlnAspAlaSerThrSerSer                               225230235240                                                                   ProValGlyAspThrSerLeuAspGlyTyrTyrPheValGlnAsnArg                               245250255                                                                      HisSerGluLeuTyrLeuAspValThrAspAlaSerAsnGluAspGly                               260265270                                                                      AlaPheLeuGlnGlnTrpSerTyrSerGlyAsnGluAsnGlnGlnPhe                               275280285                                                                      AspPheGluHisLeuGluAsnAsnValTyrLysIleThrAsnLysLys                               290295300                                                                      SerGlyLysSerLeuAspValTyrAsnPheGlyThrGluAsnGlyVal                               305310315320                                                                   ArgIleGlnGlnTrpSerTyrGlyGlyAlaArgAsnGlnGlnPheThr                               325330335                                                                      ValGlnSerValGlyAspGlyTyrTyrLysIleIleProArgGlySer                               340345350                                                                      GlyLysLeuValGluValAlaAspPheSerLysAspAlaGlyGlyLys                               355360365                                                                      IleGlnGlnTrpSerAspAsnAsnGlnLeuSerGlyGlnTrpLysLeu                               370375380                                                                      IleLysSerLysSerTyrSerLysLeuIleGlnAlaGluSerTyrPhe                               385390395400                                                                   AspSerSerLysValGlnLeuGluAspThrSerAspValGlyGlyGly                               405410415                                                                      LysAsnValLysCysAspAsnGluGlyAlaTrpMetAlaTyrLysAsp                               420425430                                                                      IleAspPheProSerSerGlyAsnTyrArgIleGluTyrArgValAla                               435440445                                                                      SerGluArgAlaGlyGlyLysLeuSerLeuAspLeuAsnAlaGlySer                               450455460                                                                      IleValLeuGlyMetLeuAspValProSerThrGlyGlyTrpGlnLys                               465470475480                                                                   TrpThrThrIleSerHisThrValAsnValAspSerGlyThrTyrAsn                               485490495                                                                      LeuGlyIleTyrValGlnArgAlaSerTrpAsnIleAsnTrpIleLys                               500505510                                                                      IleThrLysIleProGluGlnSerAsnLeuAsnGlnGlyArgArgAsn                               515520525                                                                      SerLysLeuIleGlnAlaGluSerTyrPheSerTyrSerGluValGln                               530535540                                                                      LeuGluAspThrLeuAspValGlyGlyGlyLysAsnValLysCysAsp                               545550555560                                                                   LysGluGlyAlaTrpMetAlaTyrLysAspIleAspPheProSerSer                               565570575                                                                      GlySerTyrArgValGluTyrArgValAlaSerGluArgAlaGlyGly                               580585590                                                                      LysLeuSerLeuAspLeuAsnAlaGlySerIleValLeuGlyMetLeu                               595600605                                                                      AspIleProSerThrGlyGlyLeuGlnLysTrpThrThrIleSerHis                               610615620                                                                      IleValAsnValAspLeuGlyThrTyrAsnLeuGlyIleTyrValGln                               625630635640                                                                   LysAlaSerTrpAsnIleAsnTrpIleArgIleThrLysVal                                     645650                                                                         (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1979 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 194..1027                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        GAAGACAAGAGAGTTGAAACAACCATAGCCTGTTTGCTTATGACTTTCAATAAGAGATAC60                 TCGGCTTAAAGGGAACTGACTTATTCGTAGAGGCTATACCATGGATATCAGTTTCCTGGT120                TTTTATCACACTGTCTATGGCTCTCTTCTCGAGCAACGTGACAGGAACGTCAGTAACATC180                AAGGGTACGACGTGGAATAAATGAAAAACATTGTGGGTTCCGACCAGTA229                           GlyIleAsnGluLysHisCysGlyPheArgProVal                                           1510                                                                           ATTACAAGAATTATTGGTGGAGGAATAGCGACGCCTCATTCATGGCCG277                            IleThrArgIleIleGlyGlyGlyIleAlaThrProHisSerTrpPro                               152025                                                                         TGGATGGTTGGAATTTTCAAAGTAAATCCTCACCGTTTCCTTTGTGGT325                            TrpMetValGlyIlePheLysValAsnProHisArgPheLeuCysGly                               303540                                                                         GGATCTATTATTAATAAAGTCTCTGTTGTTACTGCCGCCCATTGTCTT373                            GlySerIleIleAsnLysValSerValValThrAlaAlaHisCysLeu                               45505560                                                                       GTGACGCAGTTTGGAAACAGACAGAATTATTCTATCTTCGTAAGAGTT421                            ValThrGlnPheGlyAsnArgGlnAsnTyrSerIlePheValArgVal                               657075                                                                         GGAGCCCATGACATAGACAATTCGGGTACAAATTATCAAGTGGATAAA469                            GlyAlaHisAspIleAspAsnSerGlyThrAsnTyrGlnValAspLys                               808590                                                                         GTTATTGTTCACCAGGGCTACAAACACCATTCACACTACTACGATATC517                            ValIleValHisGlnGlyTyrLysHisHisSerHisTyrTyrAspIle                               95100105                                                                       GGTTTGATTTTACTCTCGAAACCAGTCGAATACAACGACAAAATACAG565                            GlyLeuIleLeuLeuSerLysProValGluTyrAsnAspLysIleGln                               110115120                                                                      CCTGTCTGTATTCCTGAGTTCAACAAACCTCACGTGAACTTGAACAAT613                            ProValCysIleProGluPheAsnLysProHisValAsnLeuAsnAsn                               125130135140                                                                   ATTAAGGTCGTCATTACTGGTTGGGGTGTTACTGGGAAAGCTACTGAG661                            IleLysValValIleThrGlyTrpGlyValThrGlyLysAlaThrGlu                               145150155                                                                      AAACGTAACGTTCTTCGTGAATTGGAGTTGCCCGTGGTTACAAACGAA709                            LysArgAsnValLeuArgGluLeuGluLeuProValValThrAsnGlu                               160165170                                                                      CAGTGCAACAAATCTTATCAGACACTCCCATTCTCAAAATTGAACCGA757                            GlnCysAsnLysSerTyrGlnThrLeuProPheSerLysLeuAsnArg                               175180185                                                                      GGAATCACTAACGACATGATTTGTGCGGGGTTTCCGGAAGGAGGGAAA805                            GlyIleThrAsnAspMetIleCysAlaGlyPheProGluGlyGlyLys                               190195200                                                                      GATGCTTGTCAGGGCGACTCTGGTGGTCCCCTGATGTATCAGAATCCA853                            AspAlaCysGlnGlyAspSerGlyGlyProLeuMetTyrGlnAsnPro                               205210215220                                                                   ACAACAGGAAGAGTGAAAATAGTTGGAGTTGTATCATTTGGGTTCGAA901                            ThrThrGlyArgValLysIleValGlyValValSerPheGlyPheGlu                               225230235                                                                      TGTGCTCGTCCCAACTTCCCCGGTGTTTACACGCGCCTCTCGAGCTAC949                            CysAlaArgProAsnPheProGlyValTyrThrArgLeuSerSerTyr                               240245250                                                                      GTTAACTGGCTCCAGGAAATCACCTTCGGACAGTCACTCGCTTCTTTA997                            ValAsnTrpLeuGlnGluIleThrPheGlyGlnSerLeuAlaSerLeu                               255260265                                                                      TTTGAAGTTGTACCAATATTTATACCCGAGTGAGACTGAAGATAAATATT1047                         PheGluValValProIlePheIleProGlu                                                 270275                                                                         GAAGAGAAATCTAGAATAATGTACAATATAAGAAGCCTGAAATTACTGAAATAGAAAGGC1107               GCGTGATGAGAAATACGTTTCAAATTTTATTTTTTATTAACTTTATTGTGTTTAACTATT1167               CTTTACGTGGGACATGAAATATAAATCTTTATTTCTTCTTTATATACTTTAGATTTTCAT1227               TTCATCTATCTTTATCAGTTTTGTAATGTTACTAATAATATTTCTTATGGCACGGATCGA1287               GCCTCGTGAATCACAGTAAATAATAATAATTATAAAATCACACATTATTAAAAGCAATAG1347               CATTCAGAGTGAGTAACATATAAACTTCACTATGAGTGGACTTTTTTATTCACATTTTAA1407               GTTCATTACTAACTGTTGGGAGGTCTTTATATTGTTGTATATTTATATATTAATTAGGTT1467               GGTTTAGTACATTGTTGTTAATGGTGGAATAGGGCGTAGGTTTTAAATGTGTTTGCAAAA1527               AAACAAACAAAACAAGTAATGGTGGATGATGGTTCCAAAGTAACCGAAAGAACACTTTGA1587               ACATTTTTATACAAAAATTTATGTTTTAAAATACGAGTATATACAATCGATCTCTAAGTA1647               CAAGAAAAACTGAAGTGTTCATTCAGGTTTAACAGTGCAACTTAAATCAACAGTTAGTTG1707               TTCACTAAACATTACAATTTGATCCTTTATAAACGCTAATACTGTTTAAACAGTCAGTAA1767               TAATACAGTATCATAGCATATCATATATGAAGGTATTTTAACATTCTATATACAAAGCCA1827               GAATTGAAAACGGTAATATTTTGTACGATTAGTGAATTATTGTTTTTAAGAACAAACTGG1887               TATCAAATTTAAAATATGAATCTGTGATTTAATATTTTTTACAACGTTCTAACTTACCAC1947               TTTTGTTGTGAATAAAGGTGTTTACAAATGGA1979                                           (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 278 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        GlyIleAsnGluLysHisCysGlyPheArgProValIleThrArgIle                               151015                                                                         IleGlyGlyGlyIleAlaThrProHisSerTrpProTrpMetValGly                               202530                                                                         IlePheLysValAsnProHisArgPheLeuCysGlyGlySerIleIle                               354045                                                                         AsnLysValSerValValThrAlaAlaHisCysLeuValThrGlnPhe                               505560                                                                         GlyAsnArgGlnAsnTyrSerIlePheValArgValGlyAlaHisAsp                               65707580                                                                       IleAspAsnSerGlyThrAsnTyrGlnValAspLysValIleValHis                               859095                                                                         GlnGlyTyrLysHisHisSerHisTyrTyrAspIleGlyLeuIleLeu                               100105110                                                                      LeuSerLysProValGluTyrAsnAspLysIleGlnProValCysIle                               115120125                                                                      ProGluPheAsnLysProHisValAsnLeuAsnAsnIleLysValVal                               130135140                                                                      IleThrGlyTrpGlyValThrGlyLysAlaThrGluLysArgAsnVal                               145150155160                                                                   LeuArgGluLeuGluLeuProValValThrAsnGluGlnCysAsnLys                               165170175                                                                      SerTyrGlnThrLeuProPheSerLysLeuAsnArgGlyIleThrAsn                               180185190                                                                      AspMetIleCysAlaGlyPheProGluGlyGlyLysAspAlaCysGln                               195200205                                                                      GlyAspSerGlyGlyProLeuMetTyrGlnAsnProThrThrGlyArg                               210215220                                                                      ValLysIleValGlyValValSerPheGlyPheGluCysAlaArgPro                               225230235240                                                                   AsnPheProGlyValTyrThrArgLeuSerSerTyrValAsnTrpLeu                               245250255                                                                      GlnGluIleThrPheGlyGlnSerLeuAlaSerLeuPheGluValVal                               260265270                                                                      ProIlePheIleProGlu                                                             275                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 9 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..9                                                             (D) OTHER INFORMATION: /note= "TABLE 1, PEPTIDE 1"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        SerXaaGluProLysXaaGlnLeuVal                                                    15                                                                             (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 8 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..8                                                             (D) OTHER INFORMATION: /note= "TABLE 1, PEPTIDE 2"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        ArgGluAspTyrAspGlyPheLys                                                       15                                                                             (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 7 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..7                                                             (D) OTHER INFORMATION: /note= "TABLE 1, PEPTIDE 3"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        TyrThrSerAlaArgLeuLys                                                          15                                                                             (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 5 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..5                                                             (D) OTHER INFORMATION: /note= "TABLE 1, PEPTIDE 4"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        ThrGlnPheAspLys                                                                15                                                                             (2) INFORMATION FOR SEQ ID NO:9:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 26 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..26                                                            (D) OTHER INFORMATION: /note= "TABLE 1, PEPTIDE 7"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                        MetAlaIleProSerPheArgGlyValTrpValMetPheTrpMetSer                               151015                                                                         GlyXaaAsnThrAsnTyrValXaaXaaPro                                                 2025                                                                           (2) INFORMATION FOR SEQ ID NO:10:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 6 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..6                                                             (D) OTHER INFORMATION: /note= "TABLE 1, PEPTIDE 8"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       SerAlaPheArgAsnLys                                                             15                                                                             (2) INFORMATION FOR SEQ ID NO:11:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 19 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..19                                                            (D) OTHER INFORMATION: /note= "TABLE 1, PEPTIDE 9"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                       ValPheValIleLeuAsnMetAlaIleGlyGlyAsnXaaProGlyPhe                               151015                                                                         XaaValAla                                                                      (2) INFORMATION FOR SEQ ID NO:12:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 8 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..8                                                             (D) OTHER INFORMATION: /note= "TABLE 1, PEPTIDE 10"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                       IleIleProArgGlySerGlyLys                                                       15                                                                             (2) INFORMATION FOR SEQ ID NO:13:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 13 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..13                                                            (D) OTHER INFORMATION: /note= "TABLE 1, PEPTIDE 11"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                       ValGlnLeuGluAspThrSerAspValGlyGlyGlyLys                                        1510                                                                           (2) INFORMATION FOR SEQ ID NO:14:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..11                                                            (D) OTHER INFORMATION: /note= "TABLE 1, PEPTIDE 12"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                       CysAspAsnGluGlyAlaTrpMetAlaTyrLys                                              1510                                                                           (2) INFORMATION FOR SEQ ID NO:15:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..17                                                            (D) OTHER INFORMATION: /note= "TABLE 1, PEPTIDE 13"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                       AspIleAspPheProSerSerGlyAsnTyrXaaIleGluTyrXaaVal                               151015                                                                         Ala                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 16 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..16                                                            (D) OTHER INFORMATION: /note= "TABLE 1, PEPTIDE 14"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                       LeuIleGlnAlaGluXaaTyrPheXaaTyrXaaGluValGlnLeuGlu                               151015                                                                         (2) INFORMATION FOR SEQ ID NO:17:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 8 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..8                                                             (D) OTHER INFORMATION: /note= "TABLE 1, PEPTIDE 15"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                       GluGlyAlaTrpMetAlaTyrLys                                                       15                                                                             (2) INFORMATION FOR SEQ ID NO:18:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 19 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..19                                                            (D) OTHER INFORMATION: /note= "TABLE 1, PEPTIDE 16"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                       ValArgGlyThrIleHisTrpSerThrProAspGlyAlaHisAlaHis                               151015                                                                         HisAsnArg                                                                      (2) INFORMATION FOR SEQ ID NO:19:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 25 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- feature                                              (B) LOCATION: 1..25                                                            (D) OTHER INFORMATION: /product="DNA SEQUENCE A'"                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                       CGGAGCTCGARGAYTAYGAYGGNTT25                                                    (2) INFORMATION FOR SEQ ID NO:20:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 26 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- feature                                              (B) LOCATION: 1..26                                                            (D) OTHER INFORMATION: /product="DNA SEQUENCE B'"                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                       CGGAATTCCATCCARAACATNACCCA26                                                   (2) INFORMATION FOR SEQ ID NO:21:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- feature                                              (B) LOCATION: 1..20                                                            (D) OTHER INFORMATION: /product="DNA SEQUENCE SECTION 8                        OF SPECIFICATION"                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                       AGCCACGAACCAAAGTGGCA20                                                         (2) INFORMATION FOR SEQ ID NO:22:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 24 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- feature                                              (B) LOCATION: 1..24                                                            (D) OTHER INFORMATION: /product="DNA PROBE SECTION 8 OF                        SPECIFICATION"                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                       AAACAGACCATGAGCCACGAACCA24                                                     (2) INFORMATION FOR SEQ ID NO:23:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 5 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..5                                                             (D) OTHER INFORMATION: /note= "TABLE 2, PEPTIDE 1"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                       GlyIleAsnGluLys                                                                15                                                                             (2) INFORMATION FOR SEQ ID NO:24:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 9 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..9                                                             (D) OTHER INFORMATION: /note= "TABLE 2, PEPTIDE 2"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                       XaaXaaGlyPheXaaProValIleThr                                                    15                                                                             (2) INFORMATION FOR SEQ ID NO:25:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 19 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..19                                                            (D) OTHER INFORMATION: /note= "TABLE 2, PEPTIDE 3"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                       IleIleGlyGlyGlyIleAlaThrProHisSerXaaProXaaMetVal                               151015                                                                         GlyIlePhe                                                                      (2) INFORMATION FOR SEQ ID NO:26:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 37 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..37                                                            (D) OTHER INFORMATION: /note= "TABLE 2, PEPTIDE 5"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                       ValXaaValValThrAlaAlaHisCysLeuValThrGlnPheGlyAsn                               151015                                                                         ArgGlnXaaTyrSerIlePheValArgValGlyAlaHisXaaIleXaa                               202530                                                                         AsnSerGlyThrAsn                                                                35                                                                             (2) INFORMATION FOR SEQ ID NO:27:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..11                                                            (D) OTHER INFORMATION: /note= "TABLE 2, PEPTIDE 6"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                       ValValIleThrGlyTrpGlyValThrGlyLys                                              1510                                                                           (2) INFORMATION FOR SEQ ID NO:28:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..18                                                            (D) OTHER INFORMATION: /note= "TABLE 2, PEPTIDE 7"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                       AsnValLeuArgGluLeuGluLeuProValValThrAsnGluGlnCys                               151015                                                                         XaaLys                                                                         (2) INFORMATION FOR SEQ ID NO:29:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 9 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..9                                                             (D) OTHER INFORMATION: /note= "TABLE 2, PEPTIDE 8"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                       SerTyrGlnThrLeuProPheSerLys                                                    15                                                                             (2) INFORMATION FOR SEQ ID NO:30:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 19 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..19                                                            (D) OTHER INFORMATION: /note= "TABLE 1, PEPTIDE 9"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                       LeuAsnArgGlyIleThrAsnAspMetIleCysAlaGlyPheProGlu                               151015                                                                         GlyGlyLys                                                                      (2) INFORMATION FOR SEQ ID NO:31:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..22                                                            (D) OTHER INFORMATION: /note= "TABLE 2, PEPTIDE 10"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                       AspAlaCysGlnGlyAspSerGlyGlyProLeuMetTyrGlnAsnPro                               151015                                                                         ThrThrGlyArgValLys                                                             20                                                                             (2) INFORMATION FOR SEQ ID NO:32:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 25 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- feature                                              (B) LOCATION: 1..25                                                            (D) OTHER INFORMATION: /product="DNA SEQUENCE C'"                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                       CGGAGCTCAAYGARCARTGYAAYAA25                                                    (2) INFORMATION FOR SEQ ID NO:33:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 25 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- feature                                              (B) LOCATION: 1..25                                                            (D) OTHER INFORMATION: /product="DNA SEQUENCE D'"                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                       CGGAATTCGTNGGRTTYTGRTACAT25                                                    (2) INFORMATION FOR SEQ ID NO:34:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 4 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..4                                                             (D) OTHER INFORMATION: /note= "PEPTIDE MOTIF"                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                       GlnGlnTrpSer                                                                   1                                                                              (2) INFORMATION FOR SEQ ID NO:35:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 5 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..5                                                             (D) OTHER INFORMATION: /note= "A CHAIN PEPTIDE SEQUENCE                        FROM FIG. 1"                                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                       ValLeuGlyArgThr                                                                15                                                                             (2) INFORMATION FOR SEQ ID NO:36:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 5 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..5                                                             (D) OTHER INFORMATION: /note= "SECTION OF PEPTIDE C                            SEQUENCE SET FORTH IN FIG.1"                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                       ValSerGlyArgGly                                                                15                                                                             (2) INFORMATION FOR SEQ ID NO:37:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 262 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Protein                                                          (B) LOCATION: 1..262                                                           (D) OTHER INFORMATION: /note= "BG1 A1 SEQUENCE (FIGURE 2)"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                       AlaGlyMetAsnLeuIleTrpGlnAspGluPheAsnGlyThrThrLeu                               151015                                                                         AspThrSerLysTrpAsnTyrGluThrGlyTyrTyrLeuAsnAsnAsp                               202530                                                                         ProAlaThrTrpGlyTrpGlyAsnAlaGluLeuGlnHisTyrThrAsn                               354045                                                                         SerThrGlnAsnValTyrValGlnAspGlyLysLeuAsnIleLysAla                               505560                                                                         MetAsnAspSerLysSerPheProGlnAspProAsnArgTyrAlaGln                               65707580                                                                       TyrSerSerGlyLysIleAsnThrLysAspLysLeuSerLeuLysTyr                               859095                                                                         GlyArgValAspPheArgAlaLysLeuProThrGlyAspGlyValTrp                               100105110                                                                      ProAlaLeuTrpMetLeuProLysAspSerValTyrGlyThrTrpAla                               115120125                                                                      AlaSerGlyGluIleAspValMetGluAlaArgGlyArgLeuProGly                               130135140                                                                      SerValSerGlyThrIleHisPheGlyGlyGlnTrpProValAsnGln                               145150155160                                                                   SerSerGlyGlyAspTyrHisPheProGluGlyGlnThrPheAlaAsn                               165170175                                                                      AspTyrHisValTyrSerValValTrpGluGluAspAsnIleLysTrp                               180185190                                                                      TyrValAspGlyLysPhePheTyrLysValThrAsnGlnGlnTrpTyr                               195200205                                                                      SerThrAlaAlaProAsnAsnProAsnAlaProPheAspGluProPhe                               210215220                                                                      TyrLeuIleMetAsnLeuAlaValGlyGlyAsnPheAspGlyGlyArg                               225230235240                                                                   ThrProAsnAlaSerAspIleProAlaThrMetGlnValAspTyrVal                               245250255                                                                      ArgValTyrLysGluGln                                                             260                                                                            (2) INFORMATION FOR SEQ ID NO:38:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 137 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..137                                                           (D) OTHER INFORMATION: /note= "XYN Z SEQUENCE (FIGURE 3)"                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                       AsnThrArgIleGluAlaGluAspTyrAspGlyIleAsnSerSerSer                               151015                                                                         IleGluIleIleGlyValProProGluGlyGlyArgGlyIleGlyTyr                               202530                                                                         IleThrSerGlyAspTyrLeuValTyrLysSerIleAspPheGlyAsn                               354045                                                                         GlyAlaThrSerPheLysAlaLysValAlaAsnAlaAsnThrSerAsn                               505560                                                                         IleGluLeuArgLeuAsnGlyProAsnGlyThrLeuIleGlyThrLeu                               65707580                                                                       SerValLysSerThrGlyAspTrpAsnThrTyrGluGluGlnThrCys                               859095                                                                         SerIleSerLysValThrGlyIleAsnAspLeuTyrLeuValPheLys                               100105110                                                                      GlyProValAsnIleAspTrpPheThrPheGlyValGluSerSerSer                               115120125                                                                      ThrGlyLeuGlyAspLeuAsnGlyAsp                                                    130135                                                                         (2) INFORMATION FOR SEQ ID NO:39:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 127 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..127                                                           (D) OTHER INFORMATION: /note= "XLN A SEQUENCE (FIGURE 4)"                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                       AlaAspGlyGlyGlnIleLysGlyValGlySerGlyArgCysLeuAsp                               151015                                                                         ValProAspAlaSerThrSerAspGlyThrGlnLeuGlnLeuTrpAsp                               202530                                                                         CysHisSerGlyThrAsnGlnGlnTrpAlaAlaThrAspAlaGlyGlu                               354045                                                                         LeuArgValTyrGlyAspLysCysLeuAspAlaAlaGlyThrSerAsn                               505560                                                                         GlySerLysValGlnIleTyrSerCysTrpGlyGlyAspAsnGlnLys                               65707580                                                                       TrpArgLeuAsnSerAspGlySerValValGlyValGlnSerGlyLeu                               859095                                                                         CysLeuAspAlaValGlyAsnGlyThrAlaAsnGlyThrLeuIleGln                               100105110                                                                      LeuTyrThrCysSerAsnGlySerAsnGlnArgTrpThrArgThr                                  115120125                                                                      __________________________________________________________________________ 

We claim:
 1. An isolated polypeptide having an amino acid sequence defined by amino acid residue numbers 1-654 in SEQ ID No.
 2. 2. An isolated polypeptide having an amino acid sequence defined by amino acid residue numbers 4-236 in SEQ ID No.
 2. 3. An isolated polypeptide having an amino acid sequence defined by amino acid residue numbers 247-387 in SEQ ID No.
 2. 4. An isolated polypeptide having an amino acid sequence defined by amino acid residue numbers 391-654 in SEQ ID No.
 2. 